Troubleshooting Cell Culture Media for Bioprocessing

Introduction

Cell culture media, including basal media and feeds, are key elements impacting the performance of bioprocesses. Advancements in cellular and metabolic understanding, coupled with high throughput applications, have led to evolved approaches in medium development and optimization resulting in innovative cell culture media with desired characteristics to meet specific needs.

However, challenges associated with the use of cell culture media still exist.  Examples include manufacturability, subpar stability, inconsistent performance and/or underperformance in terms of productivity and quality attributes.  In some cases, troubleshooting efforts can be guided by learned know-how but in other circumstances a systematic approach is necessary to identify the root cause.  In this Ask the Expert session, we invite you to participate in discussions on experience and lessons learned around cell culture media troubleshooting. Please submit your questions about cell culture media for bioprocessing.

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Question 1

What stability issues are different for powder media compared to liquid media? Are there any differences in compositions between them?

Powder media are typically more stable than liquid media and therefore tend to have a longer shelf life. The salts in liquid media exist in ionized forms and are subject to reaction and degradation. Another common stability issue for liquid media is precipitation of components that are less soluble or formulated at high concentrations. In contrast, stability issues of powder media can be caused by post-manufacturing physical changes such as particle settling and moisture absorption, both contributing to product heterogeneity. Compositions can be very similar between powder and liquid media except that a few compounds such as bicarbonate are typically not included in bulk powder formulations.

Question 2

Is there known correlation between media composition and glycosylation of proteins?

There are medium components known for impacting protein glycosylation through different mechanisms. For example, manganese serves as a cofactor in the glycosylation pathway and can improve glycosylation; galactose impacts glycan precursors and may improve glycosylation; high glutamine in medium formulation can cause less glycosylation due to ammonia generation.

Question 3

Are there media with carbon source different from glucose?

Yes, there are media with alternate carbon sources. For example, galactose, which has a lower metabolizing rate than glucose, may be supplemented into cell culture media to remedy the issue of high lactate generation in certain cell culture situations. Disaccharides are also commonly used in culture media for non-mammalian cells.

Question 4

What media components can reduce apoptosis and increase cell viability beyond critical density?

Apoptosis is typically caused by nutrient depletion and/or metabolite accumulation. Nutrient depletion can be prevented by proper feeding strategies. Metabolite accumulation is less of concern in a perfusion process but is often challenging to tackle in a fed-batch process. Media optimization approaches to control the accumulation of metabolites may include: reducing glutamine to control ammonia, reducing bicarbonate to modulate carbon dioxide, and use of alternate carbon sources to control lactate levels. In addition, there are reports on targeting the apoptosis pathway by the incorporation of chemicals such as caspase inhibitors or the use of silencing RNA. However, these approaches have not been incorporated into industrial processes yet.

Question 5

Is it always important to supplement media with depleted components?

Nutrient depletion causes cell apoptosis which can impact productivity levels and/or product quality therefore it is important to supplement media with depleted components. In order to achieve the best performance, a metabolically balanced feed formulation and an optimal feeding schedule should be designed and applied.

Question 6

Is chemically defined media equivalent substitution for yeastolate and other non-defined components?

For recombinant protein and monoclonal antibody production, there are commercially available chemically defined cell culture media with equivalent or better performance than non-defined media. For vaccine production applications, media with non-defined components are still widely used.

Question 7

Which serum free media is suitable for optimization of CHO and BHK cells (Cell Culture) producing recombinant blood clotting factors.

There are commercially available serum free media suitable for the optimization of recombinant blood factor expression in CHO or BHK cells. Depending on the cell type and specific molecule of interest, the media may need to be supplemented with other components (e.g., growth factors, cell protection agents, and product dependent factors). Media optimization should always be performed in conjunction with process optimization (e.g. fed-batch vs. perfusion, tuning of process parameters) to achieve optimal outcomes.

Question 8

Which composition of fatty acids is suitable for CHO and MDCK? Is cholesterol essential to grow cell line mentioned above in fed-batch culture?

Mammalian cells can synthesize fatty acids however they cannot introduce double bonds into fatty acids beyond C9. Therefore, unsaturated fatty acids, such as linoleic acid and linolenic acid, are beneficial for mammalian cell culture. Cholesterol is not essential for mammalian cells except for auxotrophic mutants (e.g.,NS0 cells).

Question 9

We are seeing a rise in ammonia in one of our CHO cultures prior to harvest. It isn’t entirely predictable so we are having trouble troubleshooting. Any suggestions?

The concentration of glutamine and/or asparagine in your cell culture media should be assessed and limited since both amino acids can be deaminated leading to ammonia as the byproduct. During late stationary phase of mammalian cell culture, cells may utilize alanine to generate pyruvate and this process also produces ammonia. Supplementation of pyruvate into the culture is a possible solution to alleviating ammonia accumulation. Additionally, the issue can potentially be addressed from a process perspective by implementing a temperature shift scheme or adjusting the harvest time.

Question 10

Can you explain how cell metabolism characteristics can be used to optimize media in CHO cells?

There are several ways of utilizing cell metabolism characteristics for medium optimization. Firstly, the metabolism rate of key medium components such as amino acids and vitamins can easily be determined by spent media analysis. The metabolic profiles can then be used for design of stoichiometrically balanced medium formulations. Since metabolic characteristics are dynamic, utilization of metabolism rates measured from both the cell growth phase and stationary production phase are best for stoichiometric balancing of basal and feed media. Secondly, cellular metabolism is an interrelated network and metabolism characteristics can guide the focus of medium optimization. One example is within the glycolysis pathway and TCA cycle. For a cell line that tends towards lactate production, medium optimization can address this issue by switching the metabolic flux towards the TCA cycle. Finally, cell metabolism characteristics can also impact the balance between biomass formation and protein production. For a cell line with high growth rate but low productivity, limiting the metabolism rates through medium optimization can force the cells work on protein production thereby improving product yield.

Question 11

What techniques can you suggest for improving media solubility for media that is difficult to get into solution?

There are approaches, such as alternating pH (either acidic or basic) and temperature (mostly warm), that can accelerate/facilitate solubilizing media compounds. However, they don’t necessarily improve solubility (maximum concentration in a saturated solution)issues. Therefore, it is important to also evaluate the composition of your media formulation to assess whether there are components that are at the limit of solubility. Additionally, chelation can be considered for stabilizing the solution of a difficult component. One can also consider exchanging a less soluble component for a biologically equivalent compound with higher solubility to overcome solubility limitations.

Question 12

Which composition of fatty acids is suitable for CHO and MDCK? Is cholesterol essential to grow cell line mentioned above in fed-batch culture?

Mammalian cells can synthesize fatty acids however they cannot introduce double bonds into fatty acids beyond C9. Therefore, unsaturated fatty acids, such as linoleic acid and linolenic acid, are beneficial for mammalian cell culture. Cholesterol is not essential for mammalian cells except for auxotrophic mutants (e.g. NS0).